This invention utilizes imaging techniques and other methods to measure the activity in a specific region of the brain in genetically modified mice to provide an indicator of the progression of Alzheimer""s disease; furthermore, it utilizes this indicator of Alzheimer""s disease progression to identify treatments with the potential to halt the progression or prevent the onset of this disorder.
Alzheimer""s disease is the most common form of memory and thinking problems (i.e., xe2x80x9cdementiaxe2x80x9d) in older people. According to one community survey, Alzheimer""s dementia affects about 10% of those over the age of 65 and almost half of those over the age of 85. As the average life span continues to increase, Alzheimer""s disease is expected to take an increasing toll on affected persons, family caregivers, and the communities in which they live.
Little by little, Alzheimer""s disease robs affected individuals of their memory, judgment, and reasoning, their ability to recognize objects and familiar faces, their language skills, and their ability to perform routine tasks. In the most severe stage of the illness, individuals may be bed-ridden, totally confused, unable to move around or communicate with others, and unable to control their bladder or bowel functions. Affected persons commonly die from the complications of infections, accidents, or malnutrition. Indeed, Alzheimer""s disease is the fourth leading cause of death in the United States.
Alzheimer""s disease takes an extraordinary toll on the affected person""s family. Family caregivers commonly feel frustrated, helpless, and physically exhausted. Half of them become clinically depressed. Many must become impoverished before the affected person is eligible for long-term residential care.
The financial impact of Alzheimer""s disease on the community continues to grow. Currently, it costs about $175,000 to care for each person with Alzheimer""s dementia. By the time today""s young adults grow old, there will be four times as many people over the age of 65, eight times as many people over the age of 85, and a smaller proportion of individuals in younger age groups who will be expected to provide care for those affected by Alzheimer""s disease.
For all of these reasons, researchers must find ways to halt the progression and prevent the onset of this devastating disorder. Scientific discoveries in the last few years have raised the hope that researchers will find effective ways to treat and prevent Alzheimer""s disease.
For instance, autopsy studies reveal several characteristic brain abnormalities (often called xe2x80x9chistopathological featuresxe2x80x9d) in patients with Alzheimer""s disease: plaques, tangles, and a loss of brain cells. Researchers have begun to characterize the cascade of molecular events that lead to plaques (e.g., the aggregation of an unusually long form of a protein called xe2x80x9cxcex2-amyloidxe2x80x9d); they have begun to characterize the molecular events that lead to the formation of tangles (e.g., the binding of phosphate molecules to a protein called xe2x80x9ctauxe2x80x9d); and they have begun to characterize the cascade of molecular events that lead to a loss of brain cells called xe2x80x9cneuronsxe2x80x9d and their connections called xe2x80x9csynapsesxe2x80x9d (e.g., inflammation, oxidation, and over-excitation of brain cells). Treatments that target one or more of these molecular events might be able to halt the progression of Alzheimer""s disease or even prevent the onset of Alzheimer""s disease altogether.
Furthermore, researchers have identified specific genes that appear to account for about half of all Alzheimer""s disease cases. Rare mutations of the amyloid precursor protein (known as xe2x80x9cAPPxe2x80x9d) gene on chromosome 21, the presenilin 1 gene on chromosome 14, and the presenilin 2 gene appear to account for about half of those individuals who develop a form of Alzheimer""s disease that runs in families and leads to the onset of memory and thinking problems prior to the age of 60 (xe2x80x9cearly-onsetxe2x80x9d). In addition to these rare mutations, the apolipoprotein E (APOE) xcex54 gene on chromosome 19 (found in about one-fourth of the population) increases the risk and hastens the onset of memory and thinking problems in about half of those who develop Alzheimer""s dementia after the age of 60. As researchers determine how the products of these genes lead to the development of Alzheimer""s disease, they will provide additional targets for treatments to halt the progression and prevent the onset of this disorder.
Finally, researchers have already identified clinical treatments that might slow the progression or delay the onset of Alzheimer""s disease. Promising treatments include, but are not limited to, anti-oxidants like vitamin E, anti-inflammatory medications like indomethacin, estrogen replacement therapy, recently developed amyloid blocking agents, and a potential Alzheimer""s disease vaccine. A treatment that delays the onset of Alzheimer""s dementia by only 5 years has the potential to cut in half the number of new cases of this devastating disease.
Despite these promising observations, one of the greatest obstacles to the discovery of Alzheimer""s disease treatments has been the absence of a marker of disease progression in laboratory animals. A marker of Alzheimer""s disease progression in laboratory animals could be used to screen pharmaceutical compounds and other treatments for their ability to halt the progression and prevent the onset of this disorder. This screening procedure could identify promising treatments, give chemists direction in the refinement or new design of promising pharmaceutical compounds, and give the pharmaceutical industry direction in selecting which treatments to test in expensive and time-consuming clinical trials. Companies need to have confidence in a potential treatment before they are willing to spend the time, effort, and millions of dollars needed to establish its efficacy and safety and merit approval from the United States Food and Drug Administration (FDA).
Capitalizing on the discovery of specific genetic risk factors for Alzheimer""s disease and refinements in genetic engineering, research groups have recently begun to produce strains of genetically modified mice that develop some of the characteristic brain abnormalities found at autopsy in persons with Alzheimer""s dementia, such as amyloid plaques. For instance, some research groups have developed transgenic mice that over express abnormal forms of human APP found in certain families with early-onset Alzheimer""s disease; some have developed transgenic mice that over express abnormal forms of the human presenilin 1 protein found in certain other families with early-onset Alzheimer""s disease; some have developed transgenic mice that over-express human the APOE xcex54 protein found in many persons with late-onset Alzheimer""s disease; and some have developed transgenic mice with a combination of genetic modifications (e.g., transgenic/knockout mice that contain a mutant form of the human APP transgene and the human APOE xcex54 transgene and lack the mouse APOE gene). Researchers continue to work on the development of genetically modified mice that could serve as a laboratory model of Alzheimer""s disease.
To date, the most promising animal models of Alzheimer""s disease have been transgenic mice who overexpress abnormal forms of APP and develop amyloid plaques (e.g., the PDAPP mice first described by Dora Games and her colleagues). While these mice represent an important advance in the effort to find Alzheimer""s disease treatments, uncertainties remain about the validity of these and other animal models and the best way to monitor disease progression in the absence of symptoms. First, transgenic mice generally lack some of the characteristic histopathological features of Alzheimer""s disease, such as tangles and neuronal loss. Second, it remains possible that amyloid plaques are insufficient to account for the clinical features of Alzheimer""s disease. Third, since amyloid is deposited in the early stages of Alzheimer""s disease and does not progress during the course of the illness, plaque formation might constitute a more valuable marker of the pre-clinical and early clinical stages of Alzheimer""s disease, than of the latter clinical stages. Finally, even though behavioral and electrophysiological evidence of memory impairment has been observed in at least one of the transgenic mouse lines, it is difficult to relate these observations to the clinical symptoms of Alzheimer""s dementia, as experienced in humans.
Because it is difficult to assess symptoms in laboratory mice, researchers need to find a marker of Alzheimer""s disease that bridges the gap between persons with the disorder and promising animal models. In accordance with the invention detailed below, brain imaging techniques provide the means to characterize the progression of Alzheimer""s disease in animals in the absence of symptoms.
Functional brain imaging techniques, such as positron emission tomography (PET), provide information about the inner workings of the human brain. Measuring the uptake of a radioactive tracer called fluorodeoxyglucose (FDG) in the brain, PET provides information about the rates of glucose metabolism in different regions of the brain and, more generally, the activity of brain cells that project to those regions. PET studies find that persons with Alzheimer""s disease have significant reductions in FDG uptake in certain regions of the brain, including posterior cingulate, parietal, temporal, and prefrontal cortex, which become more pronounced during the course of the illness. The largest reduction is in the posterior cingulate cortex, especially early in the course of the illness. This reduction becomes apparent prior to the onset of cognitive impairment in persons at genetic risk for the disorder, is correlated with the severity of cognitive impairment in persons with Alzheimer""s dementia, and may provide the earliest marker of Alzheimer""s disease progression (Reiman et al, New Engl J Med, 1996; Reiman et al, Ann Neurol, 1998; Reiman et al, Flinn Foundation Biomed Res Life Sci Symposium [abstract], 1998). While FDG uptake is preferentially reduced in the brain regions noted above, it is preferentially spared in certain other brain regions, including visual cortex, sensorimotor cortex, cerebellum, pons, and white matter regions-although there is a reduction in whole brain glucose metabolism during the latter stages of the illness. While the characteristic and progressive pattern of reductions in brain activity has been best studied using PET measurements of FDG uptake, this pattern has also been noted using other markers of regional brain activity, including, but not limited to, PET measurements of cerebral blood flow, single photon emission computed tomographic (SPECT) measurements of cerebral blood flow, magnetic resonance imaging measurements of cerebral blood flow and blood volume, and magnetic resonance spectroscopic measurements of N-acetyl aspartic acid.
The invention utilizes measurements of brain activity in posterior cingulate cortex as an indicator of Alzheimer""s disease progression in mice engineered to contain a genetic risk factor for this disorder. The method provides the capacity to study factors that cause or influence the development of Alzheimer""s disease. It provides the capacity to screen existing, modified, or newly developed pharmaceutical compounds and other clinical treatments for their ability to halt the progression or prevent the onset of this disorder.
In accordance with one embodiment of the present invention, treated and untreated mice of sufficiently advanced age (e.g., between 6-18 months of age) are compared in terms of brain activity. More particularly, in accordance with the invention, mice containing one or more genes known to cause or to increase the risk of Alzheimer""s disease are treated for a designated time during the course of their relatively short lives, while another group of genetically comparable mice (studied previously or in random order) are followed for the same age interval in the absence of treatment or with placebo treatment only. The number of mice in each group is comparable and sufficient to detect statistically significant effects of treatment on an indicator of Alzheimer""s disease progression. Potential treatments include, but are not limited to, pharmaceutical compounds, dietary supplements, gene therapies, vaccines, and behavioral or environmental therapies. At the end of the treatment period, an imaging technique is used to measure activity (e.g., FDG uptake) in different regions of the brain. Measurements of brain activity in the posterior cingulate cortex in older untreated animals may be compared to that in untreated younger animals to characterize the extent of Alzheimer""s disease progression in the absence of treatment. Further, measurements of brain activity in older treated animals may be compared to that in untreated younger animals to characterize the extent of Alzheimer""s disease progression following treatment. Moreover, posterior cingulate activity in older treated animals may be compared to that in older untreated animals to characterize the effects of treatment on Alzheimer""s disease progression. In accordance with a preferred embodiment of the present invention, if older treated mice exhibit a higher level of brain activity in the posterior cingulate region than do older untreated mice, then a potentially efficacious treatment for Alzheimer""s disease is identified.
One technique to characterize the age-related reductions in posterior cingulate activity is known as xe2x80x9cFDG autoradiography.xe2x80x9d Using this technique, a radioactive form of the compound FDG is injected into an abdominal region of the mouse known as the peritoneum in an extremely small (xe2x80x9ctracerxe2x80x9d) dose. After a short period of time (e.g., about 45 minutes), the mouse is sacrificed, the brain is extracted and frozen, and the brain is then cut into extremely thin sections. The sections are mounted onto slides and exposed to film in order to develop images of FDG uptake. After the posterior cingulate cortex and other brain regions are identified by a person who is unaware of the animal""s treatment status, measurements in these regions are recorded and normalized for the variation in absolute brain measurements. Reductions in posterior cingulate FDG uptake in older untreated transgenic mice (in comparison with aged non-transgenic mice or young transgenic mice) provide an indicator of Alzheimer""s disease. An attenuation in posterior cingulate FDG uptake reductions in older treated mice (in comparison with the untreated mice) is an indicator of treatment efficacy, that is, a promising treatment of Alzheimer""s disease in humans. A treatment which reduces (or reverses) the decline in posterior cingulate FDG uptake, and is thought to be safe and tolerable, would be considered a promising candidate for clinical trials. A treatment which reduces the decline in posterior cingulate FDG uptake in addition to reducing the progression of other histopathological changes (e.g., plaque formation) would be considered an especially promising candidate for clinical trials.
In accordance with the invention, several functional brain imaging techniques and other methods may be suitable for characterizing the progression of Alzheimer""s disease in transgenic mice and other promising animal models: those that produce relatively high-resolution (sharp) images reflecting the activity of neurons that project to the posterior cingulate cortex, the density of synapses in the posterior cingulate cortex, or the activity or density of surrounding (xe2x80x9cglialxe2x80x9d) brain cells in this region. Imaging techniques may include, but are not limited to, certain autoradiographic and histochemical methods (which produce relatively sharp images, but require animal sacrifice) and positron emission tomography (PET), single emission computed tomography (SPECT), and magnetic resonance imaging (PET) systems especially designed for the study of small animals (which produce relatively blurry images, but permit researchers to use each animal as its own control as the living brain is studied on more than one occasion). Useful indicators of the processes noted above may include, but are not limited to, to markers of cerebral metabolism, blood flow, blood volume, mitochondria activity (e.g., cytochrome oxidase activity), other markers of synaptic density or activity, and other neurotransmitter or nerve receptor measurements that could reflect such activities. Images may be analyzed using preselected regions-of-interest or using computer algorithms that stack brain sections into a three-dimensional volume, transform each brain into the same orientation, size, and shape, average functional images from different brains, and create statistical maps of significant reductions in regional brain activity in the presence or absence of treatment.
The method of the present invention provides an indicator of disease progression in animal models that has been shown to correspond with the course of Alzheimer""s disease in cognitively normal persons at genetic risk for Alzheimer""s disease and persons with Alzheimer""s dementia. For this reason, it provides a useful way to study treatments designed to prevent Alzheimer""s disease and also treatments to attenuate or reverse the progression of Alzheimer""s dementia in humans. Furthermore, it provides an indicator of Alzheimer""s disease progression independent of the histopathological features which may or may not be present in the laboratory animals and may or may not be necessary or sufficient to cause Alzheimer""s disease.
To date, the invention, monitoring reductions in posterior cingulate activity, appears to provide the best way to follow the progress of Alzheimer""s disease in the absence of symptoms: first, to screen promising treatments in laboratory animals; subsequently, to investigate the ability of treatments to prevent (or delay onset of) Alzheimer""s disease in cognitively normal persons at genetic risk for Alzheimer""s disease without having to wait many years to determine whether or when treated persons go on to develop symptoms.